FIELD OF THE INVENTION
[0001] The present disclosure relates generally to pharmaceutical compositions. More specifically, the disclosure is directed to a pharmaceutical composition for management of epilepsy comprising imatinib, or a pharmaceutically acceptable salt thereof.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Epilepsy is a neurological disease characterized by spontaneous seizures induced oxidative stress-mediated neuronal impairment. The prevalence of the disease is much higher in older age patients than younger ones, estimated to be 86 per 100,000 per year in older people compared to about 23–31 per 100,000 in people aged 30 to 59 years (Beghi E. The epidemiology of epilepsy. Neuroepidemiology. 2020, 54(2), 185-91). Sudden unexpected death in epileptic patients with status epilepticus is unpredictable and frequently associated with neuronal changes including loss of neurons, gliosis as well as increase in inflammatory mediators indicating damage in the brain (Shorvon S, Tomson T. Sudden unexpected death in epilepsy. The Lancet. 2011, 378(9808), 2028-38). Oxidative stress is a feature of neuronal organelle damages like mitochondrial and endoplasmic stress-mediated neuronal apoptotic death (Li J, Zhang D, Brundel BJ, Wiersma M. Imbalance of ER and Mitochondria Interactions: Prelude to Cardiac Ageing and Disease?. Cells. 2019, 8(12), 1617). In epilepsy, changes in the neuronal configuration are recognized by the alterations in receptors functioning which stimulates the neuroimmune system, promoting the migration of microglial cells which release the inflammatory mediators that cause oxidative stress and apoptotic neuronal death (Vezzani A, Friedman A, Dingledine RJ. The role of inflammation in epileptogenesis. Neuropharmacology. 2013, 69, 16-24; Sanz P, Garcia-Gimeno MA. Reactive glia inflammatory signaling pathways and epilepsy. International Journal of Molecular Sciences. 2020, 21(11), 4096). Thus, prolonged seizures resulting in mitochondrial and endoplasmic dysfunctioning mediated neuronal death is regulated by downstream activation of cell signaling pathway networks. The activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 mitogen-activated protein kinase pathways seem to be involved in mediating neuronal death under the stressful condition of prolonged spontaneous seizures in the epileptic brain. Therefore, targeting modulators of neuronal cell death or excitotoxicity in epilepsy may provide a valuable neuroprotective strategy in the management of epilepsy and providing a better understanding of the molecular mechanism involved in epileptogenesis (Vitaliti G, Pavone P, Marino S, Saporito MA, Corsello G, Falsaperla R. Molecular mechanism involved in the pathogenesis of early-onset epileptic encephalopathy. Frontiers in molecular neuroscience. 2019, 12, 118).
[0004] Protein kinases play a key role in neuronal activation processes and integrating downstream signaling pathways by protein-protein interaction (Schenk PW, Snaar-Jagalska BE. Signal perception and transduction: the role of protein kinases. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 1999 Feb 9;1449(1):1-24; Liu J, Schenker M, Ghiasvand S, Berdichevsky Y. Kinase inhibitors with antiepileptic properties identified with a novel in vitro screening platform. International journal of molecular sciences. 2019, 20(10), 2502). Likewise, c-Abl tyrosine kinase is ubiquitously expressed in the nucleus and cytoplasm co-localizes with the endoplasmic reticulum (ER)-associated protein grp78 (Shaul Y. c-Abl: activation and nuclear targets. Cell Death & Differentiation. 2000 Jan;7(1):10-6; Ito Y, Pandey P, Mishra N, Kumar S, Narula N, Kharbanda S, Saxena S, Kufe D. Targeting of the c-Abl tyrosine kinase to mitochondria in endoplasmic reticulum stress-induced apoptosis. Molecular and Cellular Biology. 2001, 21(18), 6233-42). The non-receptor tyrosine kinase (c-Abl) is required for the apoptosis process by carrying the signaling of ER stress to mitochondria and releasing cytochrome C (Cyt C) into the cytosol, the cyto-c combines with Apaf1 forming a complex that activates the caspase-9 to caspase-3 and subsequently leading to DNA fragmentation (Qi X, Mochly-Rosen D. The PKCd-Abl complex communicates ER stress to the mitochondria–an essential step in subsequent apoptosis. Journal of cell science. 2008, 121(6), 804-13; Gonfloni S, Maiani E, Di Bartolomeo C, Diederich M, Cesareni G. Oxidative stress, DNA damage, and c-Abl signaling: at the crossroad in neurodegenerative diseases?. International journal of cell biology. 2012, 2012). In response to the endoplasmic stress, the c-Abl gets activated and interact with pro-apoptotic protein by contributing interaction with c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 mitogen-activated protein kinase pathways mediated neuronal death (Sanchez-Prieto R, Sanchez-Arevalo VJ, Servitja JM, Gutkind JS. Regulation of p73 by c-Abl through the p38 MAP kinase pathway. Oncogene. 2002, 21(6), 974-9). The increased expression of c-Abl observed in patients with temporal lobe epilepsy (TLE) and a rat model of epilepsy suggested the implication of c-Abl mediated apoptotic signaling in the pathogenesis of epilepsy (Chen L, Wang Z, Tang B, Fang M, Li J, Chen G, Wang X. Altered expression of c-Abl in patients with epilepsy and in a rat model. Synapse. 2014, 68(7), 306-16).
[0005] Therefore, there is a need to explore management of epilepsy by targeting the c-Abl tyrosine kinase.

OBJECTS OF THE INVENTION
[0006] An object of the present disclosure is to provide a composition for inhibition of c-Abl tyrosine kinase for treatment, prevention or amelioration of epilepsy.
[0007] An object of the present disclosure is to provide a pharmaceutical composition for management of epilepsy comprising imatinib.
[0008] Another object of the present disclosure is to provide a composition for attenuating kindling seizures induced by pentylenetetrazole (PTZ).
[0009] Yet another object of the present disclosure is to provide a composition for attenuating spontaneous seizures induced by pilocarpine (PC).

SUMMARY OF THE INVENTION
[0010] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0011] Aspects of the present disclosure aim to provide a composition to target c-Abl by using Imatinib, a selective inhibitor of c-Abl, to achieve neuroprotective effect against epilepsy, specifically PTZ and PC-induced excitotoxicity.
[0012] In an aspect, the present disclosure provides a pharmaceutical composition for targeting c-Abl tyrosine kinase for management of epilepsy comprising Imatinib, or a pharmaceutically acceptable salt thereof.
[0013] In an embodiment, the imatinib may be present in a weight percentage range of about 3% to about 5% of the composition.
[0014] In an embodiment, the composition comprises one or more pharmaceutically acceptable excipient(s). In an embodiment, the pharmaceutically acceptable excipient may be present in a weight percentage range of about 95% to about 97% of the composition.
[0015] In an embodiment, the composition attenuates kindling seizures induced by pentylenetetrazole(PTZ). In an embodiment, the composition attenuates spontaneous seizures induced by pilocarpine(PC).
[0016] In another aspect, the present disclosure provides use of a composition comprising imatinib or a pharmaceutically acceptable salt thereof, for the inhibition of c-AB1 tyrosine kinase for management of epilepsy.
[0017] In still another aspect, the present disclosure provides a method of inhibition of c-AB1 tyrosine kinase for management of epilepsy by administering to a subject in need thereof, a therapeutically effective amount of a composition comprising imatinib, or a pharmaceutically acceptable salt thereof.
[0018] Other aspects of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learnt by the practice of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
Figure 1 provides the kindled seizure severity score variation with time for PTZ-administration in mice with and without imatinib, as per an embodiment of the present disclosure. Values are expressed as Mean ± SEM, a = P<0.05 vs Vehicle Control; and b=P<0.05 vs PTZ Control.
Figure 2 provides the spontaneous recurrent seizure severity score variation with time for PC-administration in mice with and without imatinib, as per an embodiment of the present disclosure. Values are expressed as Mean ± SEM, a = P<0.05 vs Vehicle Control; and b=P<0.05 vs PC Control.
Figure 3 graphically shows the effect of Imatinib on TBARS levels in PTZ-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 4 graphically shows the effect of Imatinib on TBARS levels in PC-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 5 graphically shows the effect of Imatinib on glutathione activity in PTZ-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 6 graphically shows the effect of Imatinib on glutathione activity in PC-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 7 graphically shows the effect of Imatinib on catalase activity in PTZ-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 8 graphically shows the effect of Imatinib on catalase activity in PC-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 9 graphically shows the effect of Imatinib on IL-1ß concentration in PTZ-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 10 graphically shows the effect of Imatinib on IL-6 concentration in PTZ-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 11 graphically shows the effect of Imatinib on NF-?B concentration in PTZ-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 12 graphically shows the effect of Imatinib on TNF-a concentration in PTZ-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 13 graphically shows the effect of Imatinib on IL-1ß concentration in PC-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 14 graphically shows the effect of Imatinib on IL-6 concentration in PC-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 15 graphically shows the effect of Imatinib on NF-?B concentration in PC-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 16 graphically shows the effect of Imatinib on TNF-a concentration in PC-mediated mice with and without imatinib, as per an embodiment of the present disclosure.
Figure 17 provides the Hematoxylin and eosin staining images for (A) vehicle group; (B) pilocarpine (PC) control group; (C) pentylenetetrazole (PTZ) control group; (D) imatinib + pilocarpine (PC) and (E) imatinib + pentylenetetrazole (PTZ).

DETAILED DESCRIPTION OF THE INVENTION
[0020] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0021] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0022] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0023] In some embodiments, numbers have been used for quantifying weights, percentages, dosages, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0024] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0025] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0026] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0027] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[0028] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0029] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.
[0030] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0031] It should also be appreciated that the present disclosure can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
[0032] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0033] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0034] The term ‘therapeutically effective amount’ generally refers to the amount that when administered to a subject is sufficient to affect the treatment intended by the drug.
[0035] The term, "pharmaceutically acceptable excipient" as used herein refers to an excipient comprised of a material that is not biologically or otherwise undesirable.
[0036] The term, "subject" as used herein refers to an animal, preferably a mammal, and most preferably a human. The term "mammal" used herein refers to warm-blooded vertebrate animals of the class 'mammalia' , including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young, the term mammal includes animals such as cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig and human.
[0037] The term ‘management’ refers to treatment, amelioration and/or prevention of the condition. Management of epilepsy includes the control of epileptic seizures, generalized seizures, and status epilepticus.
[0038] Aspects of the present disclosure aim to provide a composition to target c-Abl, specifically c-Abl mediated apoptotic signaling, by using Imatinib or 4-[(4-methylpiperazin-1-yl)methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]phenyl]benzamide, a selective inhibitor of c-Abl, to evaluate its neuroprotective effect against epilepsy.
[0039] In an embodiment, the present disclosure provides a pharmaceutical composition for targeting c-Abl tyrosine kinase for management of epilepsy comprising Imatinib, or a pharmaceutically acceptable salt thereof.
[0040] In an embodiment, the pharmaceutically acceptable salt may be selected from lactate, laurate, chloride, bromide, phosphate, sulfate, tosylate, mesylate, citrate, or combinations thereof.
[0041] In an embodiment, the imatinib may be present in a weight percentage range of about 3% to about 5% of the composition.
[0042] In an embodiment, the composition comprises one or more pharmaceutically acceptable excipient(s). In an embodiment, the pharmaceutically acceptable excipient may be present in a weight percentage range of about 95% to about 97% of the composition.
[0043] In an embodiment, the excipient may be selected from those well-known in the art. The excipient may be selected from solvents, carrier, dispersants, binders, lubricating agents, buffering agents, preservatives, stabilizers, emulsifiers, surfactants, colouring agents, flavouring agents, sugars, diluents, or combinations thereof. However, a person of skill in the art would understand that any other excipient(s) may be employed without going beyond the spirit and scope of the disclosure.
[0044] In an embodiment, the pharmaceutically acceptable excipient may include but is not limited to natural gum, mannitol, sorbitol, sucrose, microcrystalline cellulose, phospholipids, fatty acids, metals of fatty acids, cellulosic derivatives, polyacrylates, povidones, polyvinylpyrrolidone, starch, magnesium stearate, microcrystalline cellulose, colloidal silicon dioxide, precipitated silicon dioxide, sodium bicarbonate, sodium citrate, croscarmellose sodium, crospovidone, sodium starch glyconate, stearates, talc, kaolin, alginic acid, buffers, calcium carbonate, dextrates, carbopol, carbomer, lecithin, sodium chloride, gelatine, sodium acrylate, EDTA, sodium gluconate, glycerine, potassium sorbate, ethanol, polyol, water, saline, dimethyl sulfoxide, or combinations thereof.
[0045] Neuroinflammation and Oxidative stress in epilepsy are the common features of neurodegeneration in epilepsy caused by the hypersynchronous neuronal activities in the brain. This further leads to activation of neuroinflammatory responses and apoptotic death pathways with underlying events of lipid peroxidation. Without being bound to theory, it is believed that the implication of c-Abl tyrosine kinase signaling is seen to be involved under such diverse injurious neuronal activities leading to neuronal death and involved in causing neurodegeneration in epilepsy. The selective c-Abl inhibitor Imatinib helps achieve the desired management of epilepsy.
[0046] In an embodiment, the composition attenuates epilepsy and associated symptoms. In an embodiment, the composition attenuates kindling seizures induced by PTZ. In an embodiment, the composition attenuates kindling seizures induced by PTZ for generalized tonic or clonic seizures. In an embodiment, the composition attenuates spontaneous seizures induced by PC. In an embodiment, the composition attenuates spontaneous seizures induced by PC for status epilepticus.
[0047] The composition decreases TBARS activity and increases catalase activity and glutathione activity. The composition also decreases inflammatory mediators that cause oxidative stress and apoptotic neuronal death, including but not limited to, IL-1ß concentration, IL-6, TNF-a concentration and NF?-ß concentration in the subject.
[0048] In an embodiment, the composition may be administered along with one or more therapeutically effective compound(s). In an embodiment, the composition may be in the form of a medicament.
[0049] In an embodiment, the present disclosure provides a pharmaceutical formulation for targeting c-Abl tyrosine kinase for management of epilepsy comprising Imatinib, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipient(s).
[0050] The formulation may be administered in any form known in the art and suitable for patient compliance including pills, tablets, capsules, lozenges, powders, granules, patches, suspensions, dispersions, solutions, microparticles, nanoparticles, aerosols, suppositories or sustained release particles. In an embodiment, the formulation may be delivered via injection or infusion.
[0051] In an embodiment, the formulation may preferably be administered in a dose range of about 1 mg/kg to about 500 mg/kg of body weight each day, more preferably in the range of about 1 mg/kg to about 50 mg/kg of body weight each day. The dosage may be single dosage or several dosages divided in the day. The dosage may be selected by a physician based on parameters such as patient’s weight, age, gender, severity of medical condition, and past health records.
[0052] In an embodiment, the formulation may be administered orally, cerebrally, intracerebrally, transdermally, subcutaneously, sublingually, intra-nasally, intravenously, intraperitoneally, rectally, buccally or intramuscularly. Preferably, the composition may be administered orally or intraperitoneally.
[0053] In another embodiment, the present disclosure provides use of a composition comprising imatinib or a pharmaceutically acceptable salt thereof, for the inhibition of c-AB1 for management of epilepsy.
[0054] In still another embodiment, the present disclosure provides a method of inhibition of c-AB1 for management of epilepsy by administering to a subject in need thereof, a therapeutically effective amount of a composition comprising imatinib, or a pharmaceutically acceptable salt thereof.
[0055] The management of epilepsy includes the decrease in TBARS activity and increase in catalase activity and glutathione activity. The management also involves decrease in inflammatory mediators that cause oxidative stress and apoptotic neuronal death, including but not limited to, IL-1ß concentration, IL-6, TNF-a concentration and NF?-ß concentration in the subject.
[0056] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
EXAMPLES
[0057] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
MATERIALS AND METHODS:
[0058] Experimental animals: Male Swiss albino mice weighing 25±2g maintained on a standard laboratory diet (Ashirwad Pvt. Ltd., Chandigarh, India) having free access to tap water were employed in the present study. They were housed in the departmental animal house and were exposed to 12 hr cycle of light and dark. The experiments were conducted in a semi-sound proof laboratory. The experimental protocol was approved by the institutional animal ethics committee. Care of the experimental animals was carried out as per the guidelines of the Committee for Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India.
[0059] Drug and Chemicals: The chemicals and drugs were of analytical grade and were procured from standard supplier. Pentylenetetrazole (Sigma, St. Louis, USA), imatinib (Sigma, St. Louis, USA), pilocarpine nitrate (FDC Limited, Aurangabad, India) and diazepam (Jackson Laboratories Pvt. Ltd., Amritsar, India), hyoscine butyl bromide (German Remedies Ltd., Mumbai, India)(for induction and maintenance of the disease in the mice) were dissolved in normal saline (which served as a vehicle for preparing all the drug solutions/dilutions). All drug solutions were freshly prepared before use.
[0060] EXAMPLE 1: Preparation of Composition
[0061] Preparation of the composition: A composition was prepared by dissolving 3gm of imatinib in 10 mL of normal saline.
[0062] EXAMPLE 2: Induction of Epilepsy
[0063] Experimental Protocol:
[0064] In the present study, a total of 8 groups were employed and each group comprised of 06 animals.
[0065] A. Pentylenetetrazole(PTZ) induced kindled seizure severity score assessment protocol
Group I (Vehicle control group-a): vehicle (10 ml kg-1, i.p.) every alternate day for 15 days + vehicle (10 ml kg-1 d-1, i.p.) for 16 days (day 0 to day 15).
Group II (PTZ control group): PTZ (40 mg kg-1, i.p.) every alternate day for 15 days + vehicle (10 ml kg-1 d-1, i.p.) for 16 days (day 0 to day 15).
Group III (Low dose (Imatinib) + PTZ group): PTZ (40 mg kg-1, i.p.) every alternate day for 15 days + Imatinib (1mg/kg, i.p.) for 16 days (day 0 to day 15).
Group IV (High dose (Imatinib) + PTZ group): PTZ (40 mg kg-1, i.p.) every alternate day for 15 days + Imatinib (3mg/kg, i.p.) for 16 days (day 0 to day 15).
[0066] B. Pilocarpine(PC) induced spontaneous recurrent seizure severity score assessment protocol
Group I (vehicle control group-b): vehicle (10 ml kg-1 d-1, i.p.) for the 31 days period starting 6th day after SE + vehicle (10 ml kg-1, i.p. every 20 min until the completion of 80 min period followed by diazepam (3 mg kg-1, i.p.) 40 min after last injection) on day 1.
Group II (PC control group): vehicle (10 ml kg-1 d-1, i.p.) for the 31 days period starting 6th day after SE + PC (100 mg kg-1, i.p. every 20 min until the onset of SE followed by diazepam (3 mg kg-1, i.p.) 40 min after SE) on day 1.
Group III (low dose (Imatinib) + PC group): Imatinib (1mg/kg, i.p.) for the 31 days period starting 6th day after SE + PC (100 mg kg-1, i.p. every 20 min until the onset of SE followed by diazepam (3 mg kg-1, i.p.) 40 min after SE) on day 1.
Group IV (high dose (Imatinib) + PC group): Imatinib (3mg/kg, i.p.) for the 31 days period starting 6th day after SE + PC (100 mg kg-1, i.p. every 20 min until the onset of SE followed by diazepam (3 mg kg-1, i.p.) 40 min after SE) on day 1.
[0067] 1.1 Induction of the pentylenetetrazole kindled model of epilepsy in mice: PTZ was administered in a sub-convulsant dose (40 mg/kg, i.p.) on alternate days for a total period of 15 days. The severity of kindled seizures was assessed in terms of a composite kindled seizure severity score (KSSS). Drug treatment was continued throughout the experimental procedure (once daily from day 0 to day 15 i.e. 16 days). KSSS quantification was performed 4 hrs before drug administration on the behavioral assessment day (every alternate day until day 15). The effect of Imatinib on PTZ-induced kindled seizures in mice is provided in Figure 1. Figure 1 provides the kindled seizure severity score time profile of the different groups I, II, III and IV associated with PTZ-administration. The seizure severity score was found to be maximum for PTZ-control whereas imatinib both low dose and high dose were able to significantly reduce the seizure severity score.
[0068] 1.2 Induction of the status epilepticus provoked spontaneous recurrent seizure activity in mice: Intraperitoneal application of pilocarpine (100 mg/ kg, i.p.) was repeated every 20 min until the onset of status epilepticus. A spontaneous recurrent seizure severity score (SRSSS) was recorded as a measure of the quantitative assessment of progressive development of spontaneous recurrent seizures induced after pilocarpine status epilepticus (SE). Drug treatment was started 6 days after the SE episode and was continued until day 37 (dosing was started 1 day prior to the first behavioral assessment day). Behavioral assessment was performed prior to the drug administration (every third day until day 37). The effect of Imatinib on pilocarpine-induced spontaneous recurrent seizures in mice is provided in Figure 2. Figure 2 provides the spontaneous recurrent seizure severity score time profile of the different groups I, II, III and IV associated with PC-administration. The seizure severity score was found to be maximum for PC-control, whereas imatinib both in low dose and high dose was able to significantly reduce the seizure severity score.
[0069] EXAMPLE 3: Estimation of Biochemical parameters
[0070] 3.1 Collection of sample: For biochemical estimation, the animals were sacrificed by cervical dislocation. The brains were removed and homogenized in phosphate buffer (pH 7.4, 10% w/v) using a homogenizer. The clear supernatant obtained after centrifugation at 3000 rpm for 15 min, was used to estimate various biochemical parameters. For estimation of various biochemical parameters, different methods were used, the thiobarbituric acid reactive substances (TBARS) measurement was performed as described by Ohkawa, H.; Ohishi, N.; Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical biochemistry 1979, 95(2), 351-358; reduced glutathione as described by Beutler, E.; Kelly, B.M. The effect of sodium nitrite on red cell GSH. Experientia 1963, 19(2), 96-97; catalase as described by the method in Aebi, H.Catalase “in vitro. Methods in enzymology”, 1984, 105, 121-126; and superoxide dismutase (SOD) was estimated as described by Wang, X.L.; Adachi, T.; Sim, A.S.; Wilcken, D.E.L. Plasma extracellular superoxide dismutase levels in an Australian population with coronary artery disease. Arteriosclerosis, thrombosis, and vascular biology 1998, 18(12), 1915-1921.
[0071] 3.2 Estimation of Oxidative stress: The following parameters were estimated to determine the oxidative stress in the samples.
[0072] a. Estimation of Thiobarbituric acid reactive substances (TBARS): The quantitative measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation in brain was performed by known methods. The results for TBARS activity has been provided in Figures 3 and 4. Figure 3 shows the effect of Imatinib on PTZ-mediated TBARS levels in mice and Figure 4 shows the effect of Imatinib on PC-mediated TBARS levels in mice. A reduction in TBARS was noted in both cases with imatinib.
[0073] b. Estimation of reduced glutathione(GSH): The reduced GSH content in brain tissue was estimated by method described by Beutler & Kelly, 1963. The results for GSH content analysis has been provided in Figures 5 and 6. Figure 5 shows the effect of Imatinib on PTZ-mediated glutathione activity in mice and Figure 6 shows the effect of Imatinib on PC-mediated glutathione activity in mice. An increase in GSH activity was noted in both cases with imatinib.
[0074] c. Estimation of catalase: Catalase level in brain homogenate was estimated by the method described by Aebi, 1984. The results for catalase activity analysis have been provided in Figures 7 and 8. Figure 7 shows the effect of Imatinib on PTZ-mediated catalase activity in mice and Figure 6 shows the effect of Imatinib on Pilocarpine mediated catalase activity in mice. An increase in catalase activity was noted in both cases with imatinib.
[0075] 3.3 Estimation of inflammatory mediators: The quantification of TNF-a, NF-?B, IL-6 and IL-1ß in brain was done by the help and instructions provided by Krishgen Biosystems Elisa kit.
[0076] Effect of Imatinib treatment on IL-1ß, IL-6, NF?-ß and TNF-a concentration in PTZ induced kindling model has been provided in Figures 9-12, respectively. IL-1ß concentration, IL-6 concentration, TNF-a and NF?-ß concentrations in different groups were measured by ELISA kits. Each sample was analyzed in duplicate. One way ANOVA followed by Bonferroni post hoc analysis was done (n=6/group). The IL-1ß, IL-6, NF?-ß and TNF-a which increased during epileptic seizures decreased upon administration of imatinib.
[0077] Effect of Imatinib treatment on IL-1ß, IL-6, NF?-ß and TNF-a concentration in PC-induced spontaneous recurrent seizures model has been provided in Figures 13-16, respectively. IL-1ß concentration, IL-6 concentration, TNF-a and NF?-ß concentrations in different groups were measured by ELISA kits. The IL-1ß, IL-6, NF?-ß and TNF-a values which increased during epileptic seizures decreased upon administration of imatinib.
[0078] The treatment with Imatinib at a low dose of 1 mg/kg, i.p, and high dose of 3mg/kg, i.p was screened against sub convulsive dose of PTZ (40 mg/kg; i.p.) induced kindling seizures in rodents for 16 days. Both low and high doses of Imatinib tends to be effective in attenuating epileptic seizures in rodents against PTZ kindling seizures represented by various behavioral parameters like tonic-clonic, Straub tail, Hauched back, stupor phase, wild running and jumping seizures resembling complex partial seizures, and secondarily generalized seizures. These behavioral parameters were reduced in rodents receiving treatment with Imatinib at high dose and low dose as compared to the disease control groups, the high dose of Imatinib (3mg/kg, i.p) was selected to be an effective dose as anti-epileptic in abolishing all PTZ induced seizure activities in mice as compared to the low dose (1 mg/kg; i.p.) that showed a 70 % of its effect in attenuating epileptic seizures in mice.
[0079] Both low and high dose of Imatinib were also screened for status epilepticus using the PC- model employed at a sub convulsive dose of 100 mg/kg; i.p. for 37 days. The results of Imatinib against PC-induced spontaneous recurrent seizures showed to be effective in attenuating spontaneous recurrent seizures (SRS) represented with various behavioral parameters in rodents like wet dog shake, hyperlocomotion, and stereotypes (eg, excessive grooming, rearing, and/or head movements) for the first 5 days and then accompanied with behavioral arrest, tonic-clonus phase, Straub tail, haunched back, after discharge parameters representing hippocampus area degeneration (temporal lobe epilepsy), were completely abolished with a high dose of Imatinib (3 mg/kg;i.p) in rodents. Therefore, the results confirmed the activation of c-Abl tyrosine kinase under the prolonged excessive seizures and modulating neurodegeneration by interacting with pro-apoptotic proteins regulates neuronal death.
[0080] The potential role of activation of c-Abl under chronic seizure-induced oxidative stress and DNA damage responses were evaluated by measuring oxidative stress parameters, the increased levels of antioxidant enzymes (catalase, glutathione (GSH)), and decreased TBARs levels in treatment with Imatinib, and c-Abl inhibitor as compared to the disease control groups (PTZ + Vehicle) & (PC + Vehicle). Thus, indicating the involvement of c-Abl in chronic seizure-induced oxidative stress mediating neurodegeneration in epilepsy.
[0081] Also, the ELISA results aided the interpretation of involvement of the c-Abl in the activation of NLRP3 inflammasome protein which is highly expressed in microglia cells that acts as a neuroimmune response after the diverse injurious stimuli of hyperexcitability of neurons with increased levels of NF- kB and inflammatory mediators (interleukin-1 beta (IL-1ß), interleukin-6, Tumor Necrosis Factor-alpha (TNF-a). The treatment groups with Imatinib at high dose (3 mg/kg; i.p.) was effective in suppressing the NF?-ß and decreasing the levels of pro-inflammatory mediators (IL-1 ß, IL-6 & TNF- alpha) as compared to the disease control group receiving treatment with PTZ and PC. Therefore, the results indicate the role of c-Abl in activating the NLRP3 signaling in microglia causing neuroinflammation as a result of increased chronic seizure-induced oxidative stress in epilepsy.
[0082] Furthermore, c-Abl initiates c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 mitogen-activated protein kinase cell death pathway, and activation of caspase-3 mediated apoptotic neuronal death phenomena in epilepsy. The role of c-Abl in the neuronal loss was confirmed by the histopathological evaluation of PTZ and pilocarpine-induced seizures in mice using H&E staining (refer Figure 17), the treatment control group with a high dose of Imatinib (3 mg/kg; i.p.) tends to be neuroprotective in decreasing the brain structural changes in the neurons, such as nuclear chromatin clumping, condensed cytoplasm, fragmentation of the cells for the treatment group of imatinib (1mg/kg; i.p.)+ PTZ group as well Imatinib (3 mg/kg; i.p.) + Pilocarpine group by preventing the pilocarpine-induced spontaneous recurrent seizures causing brain damage like nuclear pyknosis and shrinkage of cells as compared to the disease control groups of PTZ (40 mg kg-1, i.p.) + vehicle & Pilocarpine (100 mg kg-1, i.p.) + vehicle.
[0083] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein merely for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention and should not be construed so as to limit the scope of the invention or the appended claims in any way.

ADVANTAGES OF THE PRESENT INVENTION
[0084] The present disclosure provides a composition for management of epilepsy.
[0085] The present disclosure provides a composition that is capable of decreasing TBARS activity and increasing catalase activity and glutathione activity.
[0086] The present disclosure provides a composition that is capable of decreasing inflammatory mediators that cause oxidative stress and apoptotic neuronal death.

Claims:

1. A pharmaceutical composition for targeting c-Abl tyrosine kinase for management of epilepsy comprising Imatinib, or a pharmaceutically acceptable salt thereof.

2. The composition as claimed in claim 1, wherein the composition comprises one or more pharmaceutically acceptable excipient(s).

3. The composition as claimed in claim 1, wherein the imatinib is present in a weight percentage range of 3% to 5% of the composition.

4. The composition as claimed in claim 2, wherein the excipient is selected from solvents, carrier, dispersants, binders, lubricating agents, buffering agents, preservatives, stabilizers, emulsifiers, surfactants, colouring agents, flavouring agents, sugars, diluents, or combinations thereof.

5. The composition as claimed in claim 2, wherein the excipient is present in a weight percentage range of 95% to 97% of the composition.

6. The composition as claimed in claim 1, wherein the composition is in the form of a medicament.

7. The composition as claimed in claim 1, wherein the epilepsy is induced by pentylenetetrazole.

8. The composition as claimed in claim 1, wherein the epilepsy is induced by pilocarpine.

9. A pharmaceutical formulation for targeting c-Abl tyrosine kinase for management of epilepsy comprising Imatinib, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipient(s).

10. The formulation as claimed in claim 9, wherein the formulation is administered in the form of pills, tablets, capsules, lozenges, powders, granules, patches, suspensions, dispersions, solutions, microparticles, nanoparticles, aerosols, suppositories or sustained release particles.